Accessing the specialized metabolome of actinobacteria from the bulk soil of Paullinia cupana Mart. on the Brazilian Amazon: a promising source of bioactive compounds against soybean phytopathogens.

The Amazon rainforest, an incredibly biodiverse ecosystem, has been increasingly vulnerable to deforestation. Despite its undeniable importance and potential, the Amazonian microbiome has historically received limited study, particularly in relation to its unique arsenal of specialized metabolites. Therefore, in this study our aim was to assess the metabolic diversity and the antifungal activity of actinobacterial strains isolated from the bulk soil of Paullinia cupana, a native crop, in the Brazilian Amazon Rainforest. Extracts from 24 strains were subjected to UPLC-MS/MS analysis using an integrative approach that relied on the Chemical Structural and Compositional Similarity (CSCS) metric, GNPS molecular networking, and in silico dereplication tools. This procedure allowed the comprehensive understanding of the chemical space encompassed by these actinobacteria, which consists of features belonging to known bioactive metabolite classes and several unannotated molecular families. Among the evaluated strains, five isolates exhibited bioactivity against a panel of soybean fungal phytopathogens (Rhizoctonia solani, Macrophomina phaseolina, and Sclerotinia sclerotiorum). A focused inspection led to the annotation of pepstatins, oligomycins, hydroxamate siderophores and dorrigocins as metabolites produced by these bioactive strains, with potentially unknown compounds also comprising their metabolomes. This study introduces a pragmatic protocol grounded in established and readily available tools for the annotation of metabolites and the prioritization of strains to optimize further isolation of specialized metabolites. Conclusively, we demonstrate the relevance of the Amazonian actinobacteria as sources for bioactive metabolites useful for agriculture. We also emphasize the importance of preserving this biome and conducting more in-depth studies on its microbiota.

Metabologenomics analysis of Pseudomonas sp. So3.2b, an Antarctic strain with bioactivity against Rhizoctonia solani.

Introduction: Phytopathogenic fungi are a considerable concern for agriculture, as they can threaten the productivity of several crops worldwide. Meanwhile, natural microbial products are acknowledged to play an important role in modern agriculture as they comprehend a safer alternative to synthetic pesticides. Bacterial strains from underexplored environments are a promising source of bioactive metabolites. Methods: We applied the OSMAC (One Strain, Many Compounds) cultivation approach, in vitro bioassays, and metabolo-genomics analyses to investigate the biochemical potential of Pseudomonas sp. So3.2b, a strain isolated from Antarctica. Crude extracts from OSMAC were analyzed through HPLC-QTOF-MS/MS, molecular networking, and annotation. The antifungal potential of the extracts was confirmed against Rhizoctonia solani strains. Moreover, the whole-genome sequence was studied for biosynthetic gene clusters (BGCs) identification and phylogenetic comparison. Results and Discussion: Molecular networking revealed that metabolite synthesis has growth media specificity, and it was reflected in bioassays results against R. solani. Bananamides, rhamnolipids, and butenolides-like molecules were annotated from the metabolome, and chemical novelty was also suggested by several unidentified compounds. Additionally, genome mining confirmed a wide variety of BGCs present in this strain, with low to no similarity with known molecules. An NRPS-encoding BGC was identified as responsible for producing the banamides-like molecules, while phylogenetic analysis demonstrated a close relationship with other rhizosphere bacteria. Therefore, by combining -omics approaches and in vitro bioassays, our study demonstrates that Pseudomonas sp. So3.2b has potential application to agriculture as a source of bioactive metabolites.

Elicitation of Streptomyces lunalinharesii secondary metabolism through co-cultivation with Rhizoctonia solani.

The concern regarding the emergence of phytopathogens strains which are resistant to conventional agrochemicals has given support to the search for alternatives on the use of chemical pesticides in agriculture. In this context, microorganisms are considered as promising sources of useful natural compounds and actinobacteria are particularly relevant since they are known to produce several bioactive metabolites. The objective of this work was to investigate the production of secondary metabolites with antifungal activity by a strain of the actinobacteria Streptomyces lunalinharesii (A54A) under axenic conditions and in co-cultivation with the phytopathogen Rhizoctonia solani. Tests to evaluate antifungal activity of the extracts indicated the presence of diffusable molecules capable of inhibiting the growth of R. solani produced by S. lunalinharesii, especially when in the presence of the fungus during fermentation. Metabolomic analyzes allowed the putative annotation of the bioactive compounds desferrioxamine E and anisomycin, in addition to the evaluation of the metabolic profile of the isolate when grown in axenic mode and in co-cultivation, while statistical analyzes enabled the comparison of such profiles and the identification of metabolites produced in greater relative quantities in the elicitation condition. Such methodologies provided the selection of unknown features with high bioactive potential for dereplication, and several metabolites of S. lunalinharesii possibly represent novel compounds.

BIOACTIVITY OF EXTRACTS FROM SOLANACEAE AGAINST Zabrotes subfasciatus / Bioactividad de extractos de Solanaceae contra Zabrotes subfasciatus

ABSTRACT The botanical family Solanaceae has many species producing compounds with insecticidal properties, e.g. nicotine and capsaicin, which are used for pest management in agriculture. This fact provides perspectives to identify insecticidal compounds in Brazilian native species of Solanaceae. In this study, we performed a screening with 25 ethanolic extracts from 17 Solanaceae species in order to evaluate their bioactivity against the Mexican bean weevil, Zabrotes subfasciatus (Coleoptera: Chrysomelidae: Bruchinae). The bioactivity of Solanaceae ethanolic extracts (2,500 mg kg-1) was tested with residual contact bioassays. Adults of Z. subfasciatus were exposed to treated bean grains, and adult mortality, oviposition, F1 progeny and damages on grains were quantified. Most of the ethanolic extracts from Solanaceae reduced the number of eggs per sample, the egg-adult viability, the F1 progeny and the damages on bean grains promoted by Z. subfasciatus, but none of them interfered on its sex ratio. Ethanolic extract from leaves of Solanum lycocarpum A. St.-Hil promoted the most promissory effects on Z. subfasciatus. This ethanolic extracts can be a suitable alternative to control Z. subfasciatus in stored beans, mainly for small farmers and organic farmers.

RESUMEN La familia botánica Solanaceae tiene muchas especies que producen compuestos con propiedades insecticidas, e.g. nicotina y capsaicina, que se utilizan para el control de plagas en la agricultura. Este hecho proporciona perspectivas promisorias para identificar compuestos insecticidas en especies nativas brasileñas de Solanaceae. En el presente estudio se llevó a cabo un cribado con 25 extractos etanólicos de 17 especies de Solanaceae con el propósito de evaluar su bioactividad sobre el gorgojo pinto del frijol, Zabrotes subfasciatus (Coleoptera: Chrysomelidae: Bruchinae). La bioactividad de los extractos etanólicos se evaluó por experimentos de contacto residual. Los adultos de Z. subfasciatus se expusieron a los frijoles tratados con los extractos y se midió la mortalidad de los adultos, la ovoposición, la progenie F1 y el daño en los granos. La mayoría de los extractos etanólicos redujeron el número de huevos, la viabilidad de los huevos, la progenie y el daño en los granos, pero ninguno de ellos interfirió en la proporción sexual de los insectos. El extracto etanólico de las hojas de Solanum lycocarpum A. St.-Hil promovió los efectos más prometedores sobre Z. subfasciatus. Este extracto puede ayudar a controlar a Z. subfasciatus en frijoles almacenados, principalmente a los pequeños agricultores y a los agricultores orgánicos.

Scorpionicidal activity of secondary metabolites from Paecilomyces sp. CMAA1686 against Tityus serrulatus.

INTRODUCTION: Urban pests pose enormous risks to human health. Control initiatives are carried out in regions of high infestation and incidence of accidents caused by scorpions OBJECTIVE: In this study, we aimed to analyze the anti-scorpionic activity of fungal isolates obtained from a cemetery in Brazil. MATERIALS AND METHODS: A total of thirteen fungi were subjected to a bioassay test against Tityus serrulatus, and the two isolates with the highest scorpionicidal activity were selected for molecular identification through sequencing of the ITS DNA hypervariable region and large-scale cultivation on liquid medium for secondary metabolite extraction. The crude extracts were partitioned by solid-phase extraction, and the resulting purified extracts were tested for anti-scorpionic activity. The extracts from one of the isolates presented better results and were submitted to UPLC-MS/MS. The metabolomics data were submitted to GNPS website for Molecular Networking and MASST searches. We also performed a MolNetEnhancer analysis to identify the chemical classes of the molecules found in the samples. RESULTS: The most promising fungal isolate was identified as Paecilomyces sp. CMAA1686 which has 98% of similarity to Paecilomyces formosus. The sub-fractions C and D had the best activity against the scorpions (54 and 32% mortality, respectively). Molecular Networking and MolNetEnhancer revealed a range of molecular classes in our extracts that are known to include bioactive metabolites from Paecilomyces species. CONCLUSIONS: The scorpionicidal activity of Paecilomyces sp. CMAA1686 and its secondary metabolites may provide new alternative compounds for biological and chemical control of scorpions from the species T. serrulatus. Paecilomyces sp. CMAA1686 is an isolate that has great potential for isolation of secondary metabolites.

The bacterial and fungal communities associated with Anthurium ssp. leaves: Insights into plant endemism and microbe association.

It is known that different plant species select specific microbes to live inside their tissues in a process determined by the host genotype, phenotype and geographic location, which can introduce discussion on plant endemism and the assembly of specific microbial communities. Herein, we report the results of an investigation relating the geographic distribution of plant species and the composition of microbial communities associated with plant hosts. The bacterial and fungal community associated with Anthurium plant leaves was mapped to assess the diversity and ecology of the endophytic community associated with Anthurium spp. collected on islands and on the Brazilian mainland. Twenty-six Anthurium specimens were surveyed, distributed throughout the São Paulo state coastline, including Alcatrazes Island, some coastal islands and distinct mainland environments. Bacterial and fungal endophytes were obtained from the leaves of A. alcatrazense, A. loefgrenii, A. penthaphyllum, A. urvellianum and A. intermedium and subjected to massive bacterial 16S rRNA and fungal ITS sequencing. The results indicated that A. alcatrazense, endemic to Alcatrazes Island, hosted a specific bacterial community structure, while its fungal community was similar to that of Anthurium species from other locations. Betaproteobacteria showed a high differential occurrence in A. alcatrazense. Some groups of fungi were found mainly inhabiting A. loefgrenii plants. While Alphaproteobacteria, Gammaproteobacteria, Actinobacteria and Sordariomycetes, Dothiodeomycetes and Tremellomycetes composed the core microbial community among Anthurium plants. The results suggest crucial role for the bacterial communities to endemic plants, while endophytic fungal diversity is less specifically distributed among endemic and nonendemic plant species.

Gloeosporiocide, a new antifungal cyclic peptide from Streptomyces morookaense AM25 isolated from the Amazon bulk soil.

Actinobacteria are known by their ability to produce several antimicrobial compounds of biotechnological interest. Thus, in this study, we isolated and identified by partial 16S RNA sequencing ∼100 actinobacteria isolates from guarana (Paullinia cupana) bulk soil. Besides, we isolated from the actinobacteria Streptomyces morookaense AM25 a novel cyclic peptide, named gloeosporiocide, molecular formula C44H48N11O7S3 (calculated 938.2901), and characterized by the presence of cyclized cysteins to form three thiazols. The novel compound had activity against the plant pathogen Colletotrichum gloeosporioides, assayed by the paper disk diffusion method (42.7% inhibition, 0.1 mg disk-1) and by the microdilution assay (1.25 g L-1). Our results reveal the potential of the actinobacteria from the Amazon rhizospheric soils as biocontrol agents as well as producers of new compounds with antifungal activity. Thus, this work constitutes a step forward in the development of the biotechnology of actinobacteria in the production of compounds of agronomic interest.

YTOX: a rapid toxicity test based on the dehydrogenase activity of Saccharomyces cerevisiae for detection of contaminants in water samples.

A simple generic toxicity method (test) is proposed using baker's yeast to mediate the reduction of the colourless triphenyltetrazolium chloride (TTC) to red, 1,3,5-triphenyl formazan, which can be extracted by dimethyl sulfoxide (DMSO), enabling the identification of reducible toxic compounds (e.g. cadmium, fipronil) in water for consumption.

Secondary metabolites produced by endophytic fungi: novel antifungal activity of fumiquinone B

Fungi are present in the most diverse environments including the interior of plant tissues, living as endophytes without causing apparent damage. These endophytes are producers of secondary metabolites, also known as natural products, such as fungicides. Here, we evaluated the ethyl acetate fractions obtained from endophytic fungiisolated from plants in the genus Begonia. The fractions were submitted to inhibitorytest against the plant pathogens Diaporthe phaseolorum and Colletotrichum gloeosporioides. From the 88 ethyl acetate fractions evaluated, 14.7 % inhibited C. gloeosporioidesand 11.3 %inhibited D. phaseolorum. One fungal isolate displaying an active fraction was selected for chemical investigation. The fungus identified as Neopestalotiopsissp., produced a compound that was active against D. phaseolorum, with a MIC of 312 μg mL-1(1,695.3 μM). The compound was identified by mass spectrometry and 1H NMR as the known compound fumiquinone B. The results highlight that the endophytes are capable of producing compounds that may be used to control plant pathogens. The compound fumiquinone B is reported for the first time as an antifungal agent against D. phaseolorum, a relevant plant pathogen worldwide. This is also the first report of the production of fumiquinone B by the genus Neopestalotiopsis.

Comparison of the antioxidant property of acerola extracts with synthetic antioxidants using an in vivo method with yeasts.

In this study, we compared the antioxidant activity of ripe and unripe acerola extracts with synthetic antioxidants (BHA and BHT). This activity was assessed by classical approaches (DPPH and ABTS) and by an in vivo method using yeasts. Acerola extracts contain phenolic compounds and ascorbic acid that exhibit radical scavenger capacity and reducing power. The results obtained with yeasts revealed that the acerola extracts and BHT either acted as antioxidants or presented no activity depending on the nature of the oxidant molecule used. BHA decreased yeast resistance to oxidative treatments and also showed deleterious effects even when oxidative treatments were not applied. The unripe acerola was the most efficient antioxidant in the in vitro experiments but not necessarily in the in vivo assays, showing the weakness of in vitro systems in predicting antioxidant responses for biological purposes. BHA presented cell damaging effects even in the absence of oxidizing reagents.

Non-ribosomal peptides produced by Brazilian cyanobacterial isolates with antimicrobial activity.

Cyanobacterial strains isolated from terrestrial and freshwater habitats in Brazil were evaluated for their antimicrobial and siderophore activities. Metabolites of fifty isolates were extracted from the supernatant culture media and cells using ethyl acetate and methanol, respectively. The extracts of 24 isolates showed antimicrobial activity against several pathogenic bacteria and one yeast. These active extracts were characterized by Q-TOF/MS. The cyanobacterial strains Cylindrospermopsis raciborskii 339-T3, Synechococcus elongatus PCC7942, Microcystis aeruginosa NPCD-1, M. panniformis SCP702 and Fischerella sp. CENA19 provided the most active extracts. The 50 cyanobacterial strains were also screened for the presence of non-ribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) genes and microcystin production. Putative fragment genes coding for NRPS adenylation domains and PKS keto-synthase domains were successfully PCR amplified from 92% and 80% of cyanobacterial strains, respectively. The potential therapeutical compounds siderophores were detected in five cyanobacterial isolates. Microcystin production was detected by ELISA test in 26% of the isolates. Further a protease inhibitor substance was detected by LC-MS/MS in the M. aeruginosa NPLJ-4 extract and the presence of aeruginosin and cyanopeptolin was confirmed by PCR amplification using specific primers, and sequenced. This screening study showed that Brazilian cyanobacterial isolates are a rich source of natural products with potential for pharmacological and biotechnological applications.

Anti-inflammatory activity of Dasyphyllum brasiliensis (Asteraceae) on acute peritonitis induced by beta-glucan from Histoplasma capsulatum.

The tea prepared from leaves and thorns of Dasyphyllum brasiliensis (Asteraceae) is used in the traditional medicine in Brazil for the treatment of oral and oropharyngeal diseases. In this study, we investigated the anti-inflammatory activity of this plant. The aqueous crude extract (ACE), the methanol-water (MeOH-H(2)O) fraction obtained by solvent partition and its fractionation products were evaluated for their anti-inflammatory activities on acute peritonitis induced by beta-glucan from the cell walls of Histoplasma capsulatum. The antiedematogenic activity was also tested using the carrageenan-induced paw edema assay in mice. Oral administration of 100 and 300mg/kg of the ACE in mice caused a significant reduction of neutrophil and eosinophil recruitment in the acute peritonitis assay. In addition, ACE at 300mg/kg inhibited the number of mononuclear cells recruitment. The MeOH-H(2)O fraction and its fractionation products (all at 100mg/kg) also presented anti-inflammatory activities, confirmed by the inhibition of cells recruited to the peritoneal cavity. ACE at 100mg/kg did not show any significant reduction of the edema in the mice paw injected with carrageenan. These data together suggest that Dasyphyllum brasiliensis presents significant anti-inflammatory activity, thus supporting the popular use of the tea in the treatment of inflammatory diseases.